Towards the Establishment of a Porcine Model to Study Human Amebiasis

BACKGROUND: Entamoeba histolytica is an important parasite of the human intestine. Its life cycle is monoxenous with two stages: (i) the trophozoite, growing in the intestine and (ii) the cyst corresponding to the dissemination stage. The trophozoite in the intestine can live as a commensal leading to asymptomatic infection or as a tissue invasive form producing mucosal ulcers and liver abscesses. There is no animal model mimicking the whole disease cycle. Most of the biological information on E. histolytica has been obtained from trophozoite adapted to axenic culture. The reproduction of intestinal amebiasis in an animal model is difficult while for liver amebiasis there are well-described rodent models. During this study, we worked on the assessment of pigs as a new potential model to study amebiasis. METHODOLOGY/PRINCIPAL FINDINGS: We first co-cultured trophozoites of E. histolytica with porcine colonic fragments and observed a disruption of the mucosal architecture. Then, we showed that outbred pigs can be used to reproduce some lesions associated with human amebiasis. A detailed analysis was performed using a washed closed-jejunal loops model. In loops inoculated with virulent amebas a severe acute ulcerative jejunitis was observed with large hemorrhagic lesions 14 days post-inoculation associated with the presence of the trophozoites in the depth of the mucosa in two out four animals. Furthermore, typical large sized hepatic abscesses were observed in the liver of one animal 7 days post-injection in the portal vein and the liver parenchyma. CONCLUSIONS: The pig model could help with simultaneously studying intestinal and extraintestinal lesion development

Characterization of S3Pvac Anti-Cysticercosis Vaccine Components: Implications for the Development of an Anti-Cestodiasis Vaccine

Background: Cysticercosis and hydatidosis seriously affect human health and are responsible for considerable economic loss in animal husbandry in non-developed and developed countries. S3Pvac and EG95 are the only field trial-tested vaccine candidates against cysticercosis and hydatidosis, respectively. S3Pvac is composed of three peptides (KETc1, GK1 and KETc12), originally identified in a Taenia crassiceps cDNA library. S3Pvac synthetically and recombinantly expressed is effective against experimentally and naturally acquired cysticercosis.Methodology/ Principal Findings: In this study, the homologous sequences of two of the S3Pvac peptides, GK1 and KETc1, were identified and further characterized in Taenia crassiceps WFU, Taenia solium, Taenia saginata, Echinococcus granulosus and Echinococcus multilocularis. Comparisons of the nucleotide and amino acid sequences coding for KETc1 and GK1 revealed significant homologies in these species. The predicted secondary structure of GK1 is almost identical between the species, while some differences were observed in the C terminal region of KETc1 according to 3D modeling. A KETc1 variant with a deletion of three C-terminal amino acids protected to the same extent against experimental murine cysticercosis as the entire peptide. on the contrary, immunization with the truncated GK1 failed to induce protection. Immunolocalization studies revealed the non stage-specificity of the two S3Pvac epitopes and their persistence in the larval tegument of all species and in Taenia adult tapeworms.Conclusions/ Significance: These results indicate that GK1 and KETc1 may be considered candidates to be included in the formulation of a multivalent and multistage vaccine against these cestodiases because of their enhancing effects on other available vaccine candidates

The transfection of BDNF to dopamine neurons potentiates the effect of dopamine D3 receptor agonist recovering the striatal innervation, dendritic spines and motor behavior in an aged rat model of Parkinson's disease.

The progressive degeneration of the dopamine neurons of the pars compacta of substantia nigra and the consequent loss of the dopamine innervation of the striatum leads to the impairment of motor behavior in Parkinson's disease. Accordingly, an efficient therapy of the disease should protect and regenerate the dopamine neurons of the substantia nigra and the dopamine innervation of the striatum. Nigral neurons express Brain Derived Neurotropic Factor (BDNF) and dopamine D3 receptors, both of which protect the dopamine neurons. The chronic activation of dopamine D3 receptors by their agonists, in addition, restores, in part, the dopamine innervation of the striatum. Here we explored whether the over-expression of BDNF by dopamine neurons potentiates the effect of the activation of D3 receptors restoring nigrostriatal innervation. Twelve-month old Wistar rats were unilaterally injected with 6-hydroxydopamine into the striatum. Five months later, rats were treated with the D3 agonist 7-hydroxy-N,N-di-n-propy1-2-aminotetralin (7-OH-DPAT) administered i.p. during 4½ months via osmotic pumps and the BDNF gene transfection into nigral cells using the neurotensin-polyplex nanovector (a non-viral transfection) that selectively transfect the dopamine neurons via the high-affinity neurotensin receptor expressed by these neurons. Two months after the withdrawal of 7-OH-DPAT when rats were aged (24 months old), immunohistochemistry assays were made. The over-expression of BDNF in rats receiving the D3 agonist normalized gait and motor coordination; in addition, it eliminated the muscle rigidity produced by the loss of dopamine. The recovery of motor behavior was associated with the recovery of the nigral neurons, the dopamine innervation of the striatum and of the number of dendritic spines of the striatal neurons. Thus, the over-expression of BDNF in dopamine neurons associated with the chronic activation of the D3 receptors appears to be a promising strategy for restoring dopamine neurons in Parkinson's disease

BDNF gene transfection by NTS-polyplex to dopamine neurons of substantia nigra pars compacta.

<p>(A) Panoramic view of the labeled TH+ neurons. (B) Amplification of the zone indicated by the frame in A. The arrowheads show the co-localization of the BDNF-flag in the TH+ neurons. This is a representative illustration of the BDNF-flag expression.</p

The combined treatment did not recover the ambulatory activity and rearing in the open field.

<p>The lesion reduced the ambulatory activity evaluated by the distance traveled (A) and frequency of rearing (B) during the first ten min in the open field. However, it did not produce bradykinesia assessed by the speed of walking (C). None of the treatments recovered the ambulatory activity or rearing (A and B). Intact rats did not showed changes either in ambulation or in rearing, throughout the experiment. The 6-OHDA lesion reduced significantly the traveled distance and frequency of rearing (A: F_{3, 6} = 73.75, <i>P</i> < 0.0001 and B: F_{3, 6} = 57.30, <i>P</i> < 0.0001, One-way repeated-measures ANOVA) but it did not produce a significant effect on bradykinesia (C: F_{3, 6} = 3.563, <i>P</i> < 0.0868, One-way repeated-measures ANOVA). *** <i>P</i> < 0.001 compared with saline-treated rats. ^# <i>P</i> = No significant difference compared with saline-treated rats (Bonferroni’s Multiple Comparison Post tests). The data are given as the mean ± SEM (<i>n</i> = 5–6 rats per group).</p

Illustration of the experimental design.

<p>Arrows with asterisk indicate an evaluation of the motor behavior, which included gait analysis, rotarod performance and ambulatory activity in the open field. Double asterisks indicate EMG recordings. Rats were 12 months old at the beginning of the experiment.</p

The combined treatment recovered normal muscle tone.

<p>The lesion increased the EMG activity of the contralateral gastrocnemius but did not affect the EMG of the ipsilateral one (A). The combined treatment normalized the EMG activity (A and B) and the effect persisted two months after the treatment (B). The D3 agonist also reduced the EMG, but the reduction was partial and occurred two months after the treatment (B). The recovery produced by the combined treatment was significant (F_{2, 6} = 5.674, <i>P</i> < 0.0414, One-way repeated-measures ANOVA). * <i>P</i> < 0.05; ** <i>P</i> < 0.01 compared with saline-treated rats (Bonferroni’s Multiple Comparison Post tests). The data are given as the mean ± SEM (<i>n</i> = 5 rats per group). A, shows a representative case.</p

Administration of the D3 agonist associated with the BDNF transfection restored motor coordination.

<p>The lesion reduced the time on the rod (A-I) and the rotarod performance (B). The combined treatment normalized the time on the rod (A-II) and the rotarod performance (B). The effect persisted two months after the treatment (A-III and B). The D3 agonist alone also recovered the motor coordination (A, B) but the rats dragged the contralateral hind limb during rod rotation (arrows in C and supplementary videos <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117391#pone.0117391.s003" target="_blank">S3 Video</a>—ROTAROD PERFORMANCE IN NORMAL SPEED and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117391#pone.0117391.s004" target="_blank">S4 Video</a>- ROTAROD PERFORMANCE IN SLOW MOTION). Saline-treated rats did not recover the rotarod performance (A, B). The recovery produced by the treatments were significant (AI: F_{3, 12} = 9.881, <i>P</i> < 0.0015; AII: F_{3, 12} = 8.879, <i>P</i> < 0.0023; AIII: F_{3, 12} = 12.49, <i>P</i> < 0.0005 and B: F_{3, 15} = 21.84, <i>P</i> < 0.0001, One-way repeated-measures ANOVA). * <i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> < 0.001 compared with saline-treated rats. ^# <i>P</i> = No significant difference compared with intact rats (Bonferroni’s Multiple Comparison Post tests). The data are given as the mean ± SEM (<i>n</i> = 5–6 rats per group). C illustrates representative cases.</p

The combined treatment recovered the nigro-striatal innervation.

<p>(A) Representative micrographs of the substantia nigra pars compacta showing TH+ neurons with the presence of rhodamine-labeled dextran amine (arrowheads), previously injected into the striatum. (B) Graph showing the percentage of rhodamine-labeled TH+ neurons with respect to the total of TH+ neurons of the same substantia nigra pars compacta, in each experimental condition. The recovery produced by the treatments was significant (F_{2, 8} = 18.34, <i>P</i> < 0.0010, One-way ANOVA). * <i>P</i> < 0.05; ** <i>P</i> < 0.01 compared with saline-treated rats (Bonferroni’s Multiple Comparison Post tests). The data are given as the mean ± SEM (<i>n</i> = 4 rats per group).</p